Infectious human papillomavirus type 31b: purification and infection of an immortalized human keratinocyte cell line

Ozbun, Michelle A.

doi:10.1099/0022-1317-83-11-2753

Cited by 47 publications

(74 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Purity of the preparation was confirmed by SDS-PAGE and Coomassie staining and by negative stain transmission electron microscopy (TEM). Titer of the prep was determined by dot blot to quantify the number of nuclease resistant pseudogenomes as previously described (19) and is expressed in viral genome equivalents (vge). PsVs produced in this manner encapsidate the H2B-GFP fusion protein and can be directly visualized by laser scanning confocal microscopy (Campos and Ozbun, unpublished observations).…”

Section: Production Of Biotinylated Gfp Labeled Hpv16 Psv Particlesmentioning

confidence: 99%

See 1 more Smart Citation

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Campos

López

et al. 2007

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of inter-laboratory data as well as quality control in clinical flow cytometry. In this paper we have described a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, that is tunable on the basis of dot-size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication (1) we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, commercial fluorescein calibration beads. The utility of the calibration beads are also herein extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dotlabeled virus particles to cells.

show abstract

Section: Production Of Biotinylated Gfp Labeled Hpv16 Psv Particlesmentioning

confidence: 99%

“…Second we established a conceptual framework for quantitative determination of the rate of viral entry into cells by using quantum dot labeled human pseudo-papillomavirus (HPV) particles. Human papillomavirus have been identified as mediators of a number of benign and malignant cancers of the skin and mucosa (19,20).…”

Section: Introductionmentioning

confidence: 99%

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Campos

López

et al. 2007

Analytical Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Thus far, researchers studying experimental HPV infections in vitro have had to rely on quantal assays such as reverse transcription (RT)-PCR techniques for the detection of newly synthesized, spliced viral RNAs following infection (26,29,34,37,38,50,51). Nevertheless, these RT-PCR assays indicate only a small percentage of exposed cells become detectably infected, suggesting experimental infections in vitro are inefficient (26,29,32,34,37,38).…”

mentioning

confidence: 99%

“…Differentiated epithelial organotypic raft tissues grown from persistently HPV-infected keratinocytes can yield concentrated virion stocks on the order of 10 8 to 10 9 viral genome equivalents per ml, corresponding to nearly 4 ϫ 10 7 viral genome equivalents per raft (37,38). Even so, it remains a challenge to investigate aspects of the early viral life cycles for a number of reasons.…”

mentioning

confidence: 99%

“…In the past decade, the organotypic (raft) tissue culture system, which supports epithelial differentiation in vitro, has greatly improved our understanding of HPV life cycles (reviewed in references 39 and 53). Furthermore, infectious virions have been cultivated in vitro from raft tissues harboring a number of high-risk HPV types, including types 16, 18, 31, and 45 and a chimeric 16/18 genome (26,29,32,34,37,38).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Human Papillomavirus Type 31b Infection of Human Keratinocytes Does Not Require Heparan Sulfate

Patterson

Smith

Ozbun

2005

J Virol

Self Cite

View full text Add to dashboard Cite

Oncogenic human papillomaviruses (HPVs) are difficult to study experimentally as they replicate at low levels in vivo. This has precluded the purification of high-risk HPV virions from in vivo lesions. Virus-like particles (VLPs) and pseudovirions from low-and high-risk HPV types can emulate various aspects of HPV virion attachment and infections. These studies suggest that HPV infection is mediated by ␣6-integrin and/or heparan-sulfonated receptors. However, whether VLPs and pseudovirions accurately reflect the infection process of HPV virions has not been verified. We generated infectious HPV31b virions from organotypic (raft) tissues and performed experimental infections in a variety of cells. Successful infection following viral attachment, internalization, and nuclear transport was assayed by detecting newly synthesized, spliced HPV transcripts using reverse transcription (RT)-PCR or RT-quantitative PCR. Most human epithelial cells were infected with HPV31b at a multiplicity of infection as low as 1 to 10 viral genome equivalents per cell. HPV31b infection was detected in other cell lines, including COS-7 monkey kidney cells, but higher viral multiplicities of infection were required. Heparin preparations of various molecular weights or heparinase I treatment of cells prevented HPV31b infection of COS-7 cells and C-33A human cervical cancer cells in reproducible and dose-dependent manners. However, these reagents were unable to block infection of human keratinocytes, including HaCaT and N/TERT-1 cells and low-passage human foreskin keratinocytes. These data suggest that HPV31b infection of human keratinocytes, the natural host cell for HPV infections in vivo, does not require a heparan-sulfonated receptor, whereas heparan sulfate is important for infection of some other cells.

show abstract

Using Organotypic (Raft) Epithelial Tissue Cultures for the Biosynthesis and Isolation of Infectious Human Papillomaviruses

Ozbun

Patterson

2014

CP Microbiology

Self Cite

View full text Add to dashboard Cite

Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitrowherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection.

show abstract

Infectious human papillomavirus type 31b: purification and infection of an immortalized human keratinocyte cell line

Cited by 47 publications

References 61 publications

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Human Papillomavirus Type 31b Infection of Human Keratinocytes Does Not Require Heparan Sulfate

Using Organotypic (Raft) Epithelial Tissue Cultures for the Biosynthesis and Isolation of Infectious Human Papillomaviruses

Contact Info

Product

Resources

About